Mounting evidence suggests that receptors, G proteins and effectors are assembled into signaling complexes in cells. We have used the techniques, known as resonance energy transfer (RET), and bimolecular fluorescence complementation (BiFC), to identify and obtain temporal information about the formation and dissociation of complexes between receptors, G proteins and effectors. [unreadable] The D4.2 dopamine receptor (D4.2R) inhibits the effector protein adenylyl cyclase (AC) by activating the inhibitory heterotrimeric G protein, Gi. Fusion proteins of D4.2R with Luc or a fluorescent protein are inactive. To create a fluorescent D4.2R that could be used in RET experiments a CCPGCC motif was added to the C-terminus (D4.2R-PGCC) or at two different positions within the third intracellular loop (D4.2R-G259C and D4.2R-G275C). The tetracysteine motif did not affect cell surface expression, ligand binding to the receptor, or agonist mediated-inhibition of AC, and FlAsH binding to this motif produced a fluorescent D4.2R that could be used as an acceptor for RET experiments. RET occurs when either D4.2R-G257C or D4.2R-G275C was co-expressed in HEK 293 cells with a Luc tagged AC (AC-Luc). There was no significant RET between AC-Luc and D4.2R PGCC. RET also occurred between the tagged D4.2R and Luc-tagged Gg. These data suggest that both G protein and AC are part of a signaling complex with D4.2R.[unreadable] The beta2-adrenergic receptor (b2AR) stimulates AC by activating the stimulatory heterotrimeric G protein, Gs. RET was observed when the b2AR was tagged with Luc (b2AR-Luc) and co-expressed with D4.2R-G259C or D4.2R-G275C suggesting that both stimulator and inhibitory receptors involved in the dual regulation of AC are present in the same signaling complex. There was no significant RET between b2AR-Luc and D4.2R-PGCC even though it was functionally indistinguishable from wild type D4.2R. This is likely a consequence of the donor and acceptor tags being to too far apart or in the wrong orientation for RET to occur. RET between Luc-tagged signaling proteins and CCPGCC-tagged D4.2R occurred in the absence of signaling, and was not affected by agonist-mediated signaling.[unreadable] BiFC was combined with RET to demonstrate the simultaneous presence of three different protein in a signaling complex. BiFC occurred when b2AR tagged with YC (b2AR-YC) and Gg tagged with YN (YN-Gg) were co-expressed in HEK 293 cells. RET occurred when AC-Luc was co-expressed with b2AR-YC and YN-Gg indicating that receptor, G protein and effector are part of the same signaling complex. Experimental evidence also supports the hypothesis that G protein-mediated signaling complexes are formed before they reach the plasma membrane. RET together with subcellular fractionation demonstrated that a complex of AC and the b2AR are present on intracellular membranes. Further, dominant-negative (DN) GTPases (Rab1 and Sar1) which block anterograde trafficking out of the endoplasmic reticulum (ER) have no effect on either b2AR/AC, Gg/AC or b2AR/Gg interactions. However, DN Rab1 and Sar1 constructs (but not DN Rabs 2, 6, 8 or 11) prevent the inclusion of Ga subunits in AC signaling complexes suggesting Ga becomes part of the complex at some point beyond the ER. In summary our data support the hypothesis that heptahelical receptors, G proteins and effectors are assembled into complexes before being transported to their target membrane, and that these complexes persist when the signal transduction pathway is activated by an agonist. This arrangement helps to explain the specificity and efficacy that is often observed during G protein-mediated signal transduction.[unreadable] Lastly, we initiated attempts to produce stable complexes of purified G proteins and recpetors for determining atomic-level resolution of the structure(s) by X-ray diffraction from crystals. This follows our previous work defining detergent conditions for mono-disperse NTS1 receptor that allow productive interaction with Gq. We are continuing this work using the invertebrate Sepia officinales rhodopsin, which is considerably more stable than NTS1 in detergent.